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`VOSSIUS & PARTNER
`l'AltNTAl-WAlTE • RECKTSANWAI.~
`StEBER1STR. 4
`81675 MUNCHEN
`
`203
`
`Antiuterotrophic effects of the pure antioestrogen ICI
`182,780 in adult female monkeys (Macaca nemestrina):
`quantitative magnetic resonance imaging
`
`M. Dukes, J.C. Waterton and A. E. Wakeling
`Cancer Research Department, ZENECA Pharmaceuticals, Meresidc, Alderley Park, Macclesfield,
`Cheshire SKJ04TG, U.K.
`
`REVISED MANUSCRIPT RECEIVED 15 February 1993
`
`ABSTRACT
`
`The antiuterotrophic efficacy of the pure antioestro-
`(~~ ICI 182,780 has been demonstrated previously by
`1gnetic resonance imaging (MRI) in ovariectomized
`oestrogen-treated monkeys (Macaca nemestrina).
`Further characterization of the effects of ICI 182, 780
`in intact adult female monkeys with normal menstrual
`cycles was undertaken to provide an indication of its
`potential actions in premenopausal women. Changes
`in the volume of uterine tissues were measured by
`MRI in early, mid and late cycle. The volume of the
`uterus varied up to fivefold between individual
`in
`individuals
`monkeys but serial observations
`provided sufficient precision to allow accurate assess(cid:173)
`ments to be made of changes in the endometrium and
`myometrium during the course of the menstrual cycle
`and following ICI 182,780 administration. In com(cid:173)
`parison with its initial size in untreated monkeys, the
`endometrium increased in volume by 60% and 125%
`in the mid and late cycle respectively. In contrast, the
`
`size of the myometrium decreased significantly. by
`16% from early to mid cycle and then recovered to
`near its initial volume in the late cycle. Treatment
`with ICI 182,780 beginning in the early part of the
`menstrual cycle prevented the growth of the uterus.
`The magnitude and duration of the response was
`dependent on whether or not ovulation occurred
`during treatment with ICI 182,780. In animals ren(cid:173)
`dered anovulatory, growth of the endometrium was
`blocked completely by ICI 182, 780 and the volume of
`the tissue declined below that present at the start of
`the menstrual cycle. Antiuterotrophic efficacy was
`significantly less in monkeys which ovulated during
`treatment with ICI 182,780. The volume of myo(cid:173)
`metrium was reduced substantially by antioestrogen
`treatment, and the difference in response between
`ovulatory and anovulatory monkeys was less marked
`than that of the endometrium.
`Journal of Endocrinology (1993) 138, 203-209
`
`~"6TRODUCTION
`
`ICI 182, 780 is a potent specific pure. anti oestrogen
`which may prove superior to conventional partial
`agonist antioestrogens in the treatment of breast
`cancer (Wakeling et al. 1991). Previous studies
`evaluated the antioestrogenic potency and efficacy of
`ICI 182,780 in ovariectomized oestrogen-treated
`monkeys (Macaca nemestrina) by magnetic resonance
`imaging (MRI) of the uterus (Waterton et al. 1991;
`Dukes et al. 1992). A daily dose of 0· 1 mg ICI
`the uterotrophic
`182,780/kg completely blocked
`action of exogenous oestradiol-17~. Single intra·
`long-acting oil-based
`injections of a
`muscular
`formulation of ICI 182,780
`in ovariectomized
`monkeys were also shown to inhibit oestrogen action
`in a dose~ependent sustained manner (Dukes et al.
`
`1992). MRI has been used to observe the uterus
`during the normal menstrual cycle in women (Demas
`et al. 1986; Haynor et al. 1986; McCarthy et al. 1986;
`Wiczyk et al. 1988; Bartoli et al. 1991) and the
`response to exogenous endocrine agents (Demas
`et al. 1986; McCarthy et al. 1986; Andreyko et al.
`1988; Zawin et al. 1990). MRI has also been used
`quantitatively to measure the changes in volume of
`the endometrium and myometrium during the normal
`menstrual cycle in M. nemestrina (Waterton et al.
`1992). The MRI studies reported here were performed
`to evaluate the antiuterotrophic effects of ICI 182,780
`in adult female monkeys with normal menstrual
`cycles. Such studies are relevant to the potential
`application of ICI 182,780 in the treatment of both
`malignant and benign oestrogen-responsive diseases
`in pre- and perimenopausal women.
`
`Journal of Endocrinology (1993) 138, 203- 209 © 1993 Journal of Endocrinology Ltd
`0022...0795/93/0138--0203 S02.00/0
`
`Printed in Great Britain
`
`MYLAN PHARMS. INC. EXHIBIT 1026 PAGE 1
`
`

`
`204 M. DU~ and others
`
`· Effects of IC/ 182.780 in adult menstruating monkeys
`
`('·
`
`MATERIALS AND METHODS
`
`Animal handling and treatment schedules
`Mature f ernale pigtail monkeys (M. nemestrina) of
`5-10 kg body weight with regular menstrual cycles
`~ere used. The.normal menstrual cycle in this species
`is 29- 32 days m length; menstrual bleeding usually
`lasts J-4 days. Daily vaginal swabs were taken to
`detect the onset of menstrual bleeding and the first
`day of menstruation was designated day 1 of the
`cycle. Where required, blood samples (2 ml) for sub(cid:173)
`sequent hormone analyses were collected daily. MRI
`examinations took place before the animals received
`their morning feed, in order to minimize artefactual
`noise associated with gut motion. Anaesthesia was
`indu~d with 0·7 mg ketamine/kg (Vetalar; Parke
`DaVls, Pontypool, Gwent, U.K.) and maintained
`with halothane (Fluothane; ICI Pharmaceuticals,
`Macclesfield, Cheshire, U.K.).
`The timing of MRI, drug treatment and blood
`sampling was scheduled by reference to the first day
`of menstruation. To provide a baseline with which to
`compare the effects of drug treatment, three MRI
`measurements of the uterus were made during each of
`two cycles in ten untreated monkeys (Waterton et al.
`1992). Images were recorded in early (days 2-5), mid
`(days 12-18) and late (days 21- 25) cycle and sched(cid:173)
`~l~ ?Y reference to the differences in cycle length in
`md1v1duals. Images were recorded at similar time(cid:173)
`points in monkeys treated with ICI 182,780 beginning
`early in the menstrual cycle (days 2-5). Two formu(cid:173)
`lations of ICI 182,780 were used: a short-acting
`propylene glycol-based solution (Fl) administered
`once daily i.m. for 25 days at a dose of O·l (n = S) or
`0:2 mg (n=5) I~I 182,780/kg, and a long-acting castor
`011-based solution (F2) given as a single i.m. injection
`of 2·5 (n=6) or 4 mg (n=6) ICI 182,780/kg.
`
`Quantitative MRI
`The method for measurement of endometrium and
`myometrium volume is described in detail elsewhere
`~aterton et al. 1991, 1992; Dukes et al. 1992).
`the study employed a Biospec 400/2.3
`~nefly,
`mstrur_nent . (Oxford Research Systems, Coventry,
`~arw1cksh1re, U.K.) to record a series of oblique
`images of the uterus. For each animal on each
`occasion, sagittal sighting images were used to deter(cid:173)
`mine the spatial co-ordinates of the uterine cervix and
`f undus. From these co-ordinates, MRI parameters
`were calculated to allow the acquisition of eight
`c~ntiguous .obliq1;1e slice images, the thickness, posi(cid:173)
`tion and onentahon of which were forced to depend
`on the vector connecting the f undus and cervix. This
`procedure aJlows reproducible presentation of the
`
`Journal of Endocrinology (1993) 138, 203- 209
`
`uterus, independent of its size, location and orienta·
`tion in the pelvis (Dukes et al. 1992; Waterton et al.
`1992). Image matrices were 256 x 256 giving an in·
`plane resolution of 0·6 mm. The appearance of the
`images (see the Plate) results from the choice of MRI
`parameters, in particular the magnetic field strength
`~f 2· 35 tesla, the echo time of 50 ms, the repetition
`time of 3 s, and suppression of the signal from fat.
`To allow comparisons between images acquired on
`different occasions, tissue volumes were measured
`rather than endometrium height or area since the
`former avoids artefacts from reposition'ing errors
`or shape changes. Endometrium and myometriurn
`volumes were obtained by pixel counting in the
`oblique images using the irregular region-of-interest
`facilities within the programs uxomr or DISNMR
`(Bruker Analytische Messtechnik GmbH, Karlsruhe,
`Germany). Only slices more fundal than the internal
`os were included in the analysis. The junction zone
`was included with the myometrium. Any contribution
`to endometrium volume from the lumen was neglected
`since, except during menstruation, this is very narrow
`and insignificant in comparison with the endornetrium
`(Dukes et al. 1992; Waterton et al. 1992).
`Although, in principle, the prescription of region(cid:173)
`of-interest can be subjective, the method employed
`minimizes errors, since the presentation of the uterus
`was the same at each examination (Waterton et al.
`1991 , 1992; Dukes et al. 1992). For each animal, the
`same observer analysed all images. Week·to-week and
`cycle-to-cycle coefficients of variation were as low as
`5% (Waterton et al. 1992).
`
`Analysis of results
`The volumes of the endometrium and myometrium in
`treated and untreated groups were calculated as the
`arithmetic mean ( ± s.E.M.) normalized by reference
`to tissue volumes in individuals at the first MRI
`measurement. Differences between group means were
`tested for significance by the unpaired Student's
`t-test.
`
`RESULTS
`
`Uterine volume in the normal menstrual cycle
`The volumes of the endometrium and myometrium
`~ere measured ?Y MRI in early, mid and late cycle
`m two cycles m each of ten different untreated
`monkeys (Text-fig. 1). The mean interval between
`repeat measurements was 4 months (range 1-9
`months). Tissue volumes varied widely between indi(cid:173)
`viduals but less so in repeat measurements in the same
`animal. Typical images recorded in one monkey at
`
`MYLAN PHARMS. INC. EXHIBIT 1026 PAGE 2
`
`

`
`Effects of IC/ 182,780 in adult menstruating monkeys
`
`M . DUKES and others 205
`
`(a)
`
`138
`
`226
`
`235
`
`238
`
`191
`
`233
`
`228
`
`219
`
`169
`
`217
`
`(b)
`
`,I
`
`(
`
`4·0
`
`3·5
`
`3·0
`
`2·5
`
`2·0
`
`1·5
`
`1·0
`
`0·5
`
`,....
`... E
`~ 0
`u
`E
`::I
`0
`> 20
`
`18
`
`16
`
`14
`
`12
`
`10
`
`8
`
`6
`
`4
`
`2
`
`0
`
`138
`
`226
`
`235
`
`238
`
`233
`191
`Animal number
`
`228
`
`219
`
`169
`
`217
`
`TEXT-FIGURE 1. (a) Endometrium and (b) myometrium volumes in untreated
`monkeys during the menstrual cycle. For each of ten animals the bars represent
`(from left) tissue volumes measured by magnetic resonance imaging in two
`separate menstrual cycles, during the early (days 2-5), mid (days 12- 18) and
`late (days 2 1-25) stages of the cycle.
`
`Journal of Endocrinology (1993) 138, 203--209
`
`MYLAN PHARMS. INC. EXHIBIT 1026 PAGE 3
`
`

`
`J
`
`206 M. DUKES and others
`
`· Effects of /Cl 182, 780 in adult menstruating monkeys
`
`200
`
`"' 4)
`E
`::>
`0
`>
`]
`Cii
`E
`~ 100
`
`0
`
`~o
`
`(b)
`
`0
`
`F2 (4·0)
`Fl (0·1)
`Untreated
`Fl (0·2)
`F2 (2·5)
`(n = 5)
`(n = 20)
`(n = 3)
`(n =4)
`(n= 3)
`TEXT· FtOU RE 2. (a) Endometrium and (b) myometrium volumes in untreated
`and ICI 182,780-treatcd anovulatory monkeys. T issue volumes at mid and late
`cycle were normalized to those recorded at the beginning of the cycle in each
`individual. From the left, values are means ± s.E.M. for untreated controls;
`monkeys receiving once daily injections of ICJ 182,780 (0·1 or 0·2 mg/kg) (Fl)
`for 25 days, beginning on cycle day 3; monkeys receiving a single injection of
`ICI 182,780 (2·5 or 4 mg/kg) (F2) on day 3.
`
`days 3 (early), 16 (mid) and 24 (late) of the cycle are
`illustrated in the Plate, figs 1- 3 and in another case at
`days 2, 16 and 23 in the Plate, figs 7-9. Analysis of the
`data showed that >75% of the variance arises from
`interanimal variability. Average coefficients of varia(cid:173)
`tion for volumes measured in different cycles were in
`the range 14-19% for the endometrium and 6-10%
`for the myometrium.
`the
`in
`in size
`increased
`The endometrium
`follicular phase of the menstrual cycle in 17 of 20
`cycles (mean increase 60%, P<O·OO I; early vs mid
`cycle volume) and this growth continued in the
`luteal phase in 18 of 20 cycles (mean increase 65%,
`P<O·OOl ; mid vs late cycle volume). Changes in the
`volume of the myometrium were smalJer but a
`consistent pattern emerged in which a decrease in
`size was observed in 18 of 20 cycles in the folJicular
`phase (mean decrease 16%, P<O·OOl; early vs mid
`cycle). In the luteal phase the volume of the
`myometrium increased in 18 of 20 cycles (mean
`100/o, P<0·02; mid vs late cycle volume). The net
`effect of the follicular phase shrinkage and luteal
`phase growth of the myometrium restored mean
`myometrium volume to near that recorded initially
`(94%, P>O·OS compared with early cycle).
`
`Journal of Endocrinology (1993) 138, 203-209
`
`Effect of ICI 182,780 on the uterus
`The effects of ICI 182,780 on the appearance of the
`uterus in MR images is shown in the Plate. Plate, figs
`4-6 and 10-12 illustrate the mid corpus of the uterus
`at early, mid and late cycle, in individuals treated
`with the rapid- (Fl) or slow- (F2) release formulations
`of ICI 182,780 respectively, compared with images
`recorded in control cycles in the same individuals
`(Plate, figs J-3 and 7- 9). The dramatic shrinkage
`of the endometrium (central high-signal zone) is
`immediately apparent; the myometrial changes are
`less obvious from visual inspection of the images but
`emerge clearly from quantitative image analysis.
`In monkeys treated with ICI 182,780, the magni(cid:173)
`tude of changes in the volume of the endometriurn
`and myometrium was dependent on whether or not
`individual monkeys were rendered anovulatory by the
`treatment. Where serum progesterone concentration
`(not shown) remained below 2 nmol/I in the second
`half of the menstrual cycle, animals were judged to be
`anovulatory; similarly where progesterone exceeded
`this value and remained elevated for at least 10 days,
`ovulation was assumed to have occurred. By this
`criterion, treatment with O· l or 0· 2 mg ICI 182, 780/kg
`daily (Fl) blocked ovulation in three or four of the
`
`MYLAN PHARMS. INC. EXHIBIT 1026 PAGE 4
`
`

`
`Effects of !CJ 182.780 in adult menstruating monkeys
`
`· M. DUKES and others 207
`
`50
`
`0
`
`Untreated
`
`F2(4·0)
`F2(2·5)
`F2(2·5)
`F2(2·5)
`Fl(0·2)
`Fl(O·l)
`Fl (O· l)
`169
`233
`219
`21 7
`174
`225
`235
`TEXT-FIGURE 3. (a) Endometrium and (b) myometrium volumes in untreated and ICI 182,780-treated ovulatory monkeys.
`Tissue volumes at mid and late cycle, normaJized to those recorded at the beginning of the cycle in each individual
`monkey, are compared with mean values ( ± s.E.M., n=20) for untreated controls. From the left, values are
`means ± s.E.M. for untreated controls; monkeys receiving once daily injections of ICI 182, 780 (O· I or O· 2 mg/kg) (Fl) for
`25 days beginning on cycle day 3; monkeys receiving a single injection of ICI 182,780 (2·5 or 4 mg/kg) (F2) on day 3.
`
`five monkeys respectively, in each dose group. Simi(cid:173)
`larly, ovulation was blocked in three or five of the six
`monkeys receiving a single injection of 2·5 or 4 mg
`JC-:1 182,780/kg (F2). There was a tendency for the
`her-dose groups to include fewer ovulatory cycles
`(
`but this was not statistically significant with the small
`group sizes employed.
`The mean changes in the volume of the endo(cid:173)
`metrium and myometrium in ICI 182,780-treated
`animals are presented in Text-figs 2 and 3. Since
`absolute tissue volumes vary widely between indi(cid:173)
`viduals (Text-fig.
`l ), the effects of ICI 182, 780
`were assessed by normalizing the volume of tissue(s)
`recorded in each animal at the mid and late cycle
`observations to those recorded at the beginning of the
`cycle, prior to treatment, and by comparison with
`similarly normalized observations in the untreated
`controls. Jn monkeys rendered anovulatory by JCI
`182,780 (Text-fig. 2), the normal follicular and luteal
`phase growth of the endometrium was blocked
`completely and tissue volume at mid and late cycle
`was significantly (P<O·OOI) less than that before
`treatment began in each of the treatment regimens.
`
`Although shrinkage of the endometrium was most
`apparent during the first half of the cycle (mean
`decrease
`- 61% compared with +600/o control,
`P<O·OOl, at mid cycle), blockade of growth was
`sustained throughout (mean - 67% compared with
`+ 125% control, P<O·OOI, at late cycle). When the
`occurrence of ovulation was accounted for, no sig(cid:173)
`nificant differences emerged between the effects of the
`different formulations and doses of ICI 182, 780, with
`the exception that the 2·5 mg dose (F2) appeared
`slightly less effective (P<O·OS) than the 4·0 mg dose in
`the second half of the cycle. The volume of the
`myometrium in ICI 182,780-treated anovulatory
`monkeys (Text-fig. 2) decreased more rapidly than in
`controls in each treatment group from early to mid
`cycle (mean - 35% compared with - 16% control,
`P <O·OOl, at mid cycle) and this decline continued in
`the second half of the cycle (mean - 54% compared
`with - 6% control, P<O·OOl, at late cycle).
`In monkeys in which treatment did not block
`ovulation (Text-fig. 3), the mean effect of ICI 182,780
`on the endometrium in the first half of the cycle
`( - 27% compared with +60% control, P<O·OI) was
`
`Journal of Endocrinology (1993) 138, 203-209
`
`MYLAN PHARMS. INC. EXHIBIT 1026 PAGE 5
`
`

`
`208 M. DUKES and others
`
`· Effects of /CI 182,780 in adult menstruating monkeys
`
`('1
`
`(
`
`I
`
`less than that seen in the anovulatory monkeys
`but not significantly (P>0·05) so. However, the
`mean response concealed wide differences between
`individual monkeys with five of the seven animals
`- 35 ± 5%) whilst the
`showing shrinkage (mean
`remaining two monkeys experienced net growth of
`the endometrium ( + 103% and + 28%). In the latter
`animals, the growth of endometrium resumed in the
`
`luteal phase of the cycle '+ 70%) at a rate similar to
`
`that in untreated monkeys ( +65%). Mean tissue
`volume remained, however, less than that in untreated
`controls at the end of the cycle (P<O·Ol), but greater
`than that in anovulatory monkeys (P<O·OOI). A
`similar pattern of change was observed in the
`myornetrium (Text.fig. 3). At mid cycle the mean
`volume of the tissue was reduced significantly
`( - 37%: P<O·Ol compared with control) and this
`effect was not different from that in anovulatory
`animals ( - 35%). However, as was the case for the
`endometrium, the myometrium did recover in the
`luteal phase ( + 18%) but not to the volume seen in
`controls. At the late cycle observation, effects of
`treatment on the myometrium were significantly less
`(P<O·Ol) in ovulatory than in anovulatory monkeys.
`
`DISCUSSION
`
`MRI provides an accurate and reproducible means of
`measuring the changes in volume of the uterus in
`response to oestrogen treatment in ovariectomized
`monkeys (Waterton et al. 1991) and has been used to
`evaluate the antiuterotrophic action of lCl 182,780
`(Dukes et al. 1992). This technique has been applied
`previously to determine the changes in volume of
`the endometrium and myometrium during the course
`of the normal menstrual cycle of M nemestrina
`(Waterton et al. 1992) and in this report to measure
`the effects of treatment with ICI 182, 780 on those
`changes.
`At the time of menstruation, uterine size, in partic·
`ular that of the endometrium, would be expected to
`reach a minimum and to increase during the follicular
`phase of the cycle and, to a lesser extent, in the luteal
`phase. To address this question, MRI measurements
`were made at early, mid and late cycle in each of two
`normal cycles in ten monkeys. In the early part of the
`cycle (days 2-5), the volume of the endometriurn and
`myometrium in individuals varied over a wide range
`(up to fivefold), but tissue volumes were much less
`variable in repetitive measurements in the same
`individual (Text-fig. l ). A clear pattern of change
`emerged during the menstrual cycle in which signifi(cid:173)
`cant growth of the endometrium was seen in both the
`follicular and luteal phases. Perhaps surprisingly, the
`mean increase in size of the endometrium was similar
`
`Journal of &docrinology (1993) 138, 203-209
`
`comparing the first and second half of the cycle.
`Oestradiol is considered to be the major mitogen for
`the uterus and it was thus anticipated that the largest
`change in the endometrium would occur in the folli(cid:173)
`cular phase of the cycle. However, a proportionately
`similar increase in the volume of the endometrium
`was seen in the luteal phase of the cycle. MRI cannot
`be used to determine whether the effects of oestrogen
`and progesterone on the size of the endometriurn
`can be attributed mainly to epithelial hyperplasia as
`opposed to hypertrophy. Changes in the size of the
`myometrium during the menstrual cycle were quanti·
`tatively much smaller than those of the endometrium
`and, in the follicular phase, qualitatively different
`(Text.fig. I). Remarkably, the myometrium decreased
`in size when the endometrium was growing, mean
`myometriurn volume being smaller in mid cycle than
`at menstruation. This may indicate that the threshold
`of sensitivity of the myometrium to oestrogens is
`higher than that of the endometrium and/or that the
`rate at which the myometrium responds to changes
`in hormonal status is less than that for the endo·
`metrium. However other mechanisms, such as
`honnone·induced changes
`in muscle
`tone, may
`influence the apparent size of the uterus and are not
`accounted for in these MRI images.
`Initial studies of the effects of ICI 182, 780 em·
`ployed a daily dosing regimen with the antioestrogen
`formulated in propylene glycol vehicle (Fl) to provide
`rapid release in vivo. It was shown previously that the
`chosen doses of ICI 182,780 (0· l-0·2 mg/kg per day)
`achieve complete and rapidly reversible blockade of
`exogenous oestrogen stimulation of the uterus in the
`ovariectomized monkeys (Dukes et al. 1992). Daily
`treatment during the menstrual cycle provided clear
`evidence that ICI 182,780 also has a substantial
`antioestrogenic effect on the uterus in intact monkeys
`(Text-figs 2 and 3). The magnitude of the anti(cid:173)
`uterotrophic effect was strongly influenced by the
`concurrent endocrine effects of ICI 182, 780, in
`particular by whether the monkeys ovulated during
`the treatment period. In anovulatory monkeys, not
`only was the normal increase in volume of the
`endometrium in the follicular phase absent, the endo·
`metrium actually decreased in size to volume com(cid:173)
`parable to those seen in ovariectomized monkeys after
`oestrogen withdrawal (Waterton et al. 1991; Dukes
`et al. 1992), indicating that treatment achieved com(cid:173)
`plete blockade of the trophic action of endogenous
`oestrogens. This blockade of oestrogen action on the
`endometrium was sustained in the second half of
`the cycle. An oestrogen withdrawaJ.Jike action on the
`myometrium was also recorded, where the normal
`small decrease in volume of the myometrium in the
`foJlicular phase was significantly amplified by ICI
`182,780, and also became similar in magnitude to that
`
`MYLAN PHARMS. INC. EXHIBIT 1026 PAGE 6
`
`

`
`Effects of /CJ 182, 780 in adult menstruating monkeys
`
`· M. DUKES and others 209
`
`seen following oestrogen withdrawal in ovariecto(cid:173)
`mized monkeys. This shrinkage of the myometrium
`continued in the luteal phase.
`The alternative oil-based long-acting formulation
`(F2) of ICI 182, 780, shown previously to sustain
`blockade of the uterotrophic action of oestradiol in
`ovariectomized monkeys for approximately 1 month
`(Dukes et al. 1992), produced effects on the uterus
`similar to those of the short-acting formulation.
`These previous pharmacological findings are entirely
`consistent with the findings in the present study with
`respect to the duration of action, the apparent dose(cid:173)
`response, and the longer sustained blockade of
`myometrial than endometrial growth. In monkeys
`rendered anovulatory, the antiuterotrophic effect of
`the 4·0 mg/kg dose was indistinguishable from that
`of the short-acting formulation. However, the lower
`2·5 mg/kg dose did not fully block the trophic action
`(',,endogenous oestrogens on the endometriurn in the
`~cond half of the cycle, but did cause progressive
`myometrial shrinkage. This provides further evidence
`that the threshold of sensitivity of the myometrium to
`oestrogens is higher than that of the endometrium.
`With both formulations, the antiuterotrophic effi(cid:173)
`cacy of ICI 182, 780 in monkeys that ovulated during
`treatment was more variable and generally smaller
`than in anovulatory animals, but still substantial in
`the majority (five of seven) of individuals (Text-figs 2
`and 3).
`Overall, of the total of 22 treated 'cycles' , 15 were
`anovulatory. With both formulations there was some
`evidence that inhibition of ovulation was dose(cid:173)
`related, but there were insufficient data to establish
`statisticalJy significant differences. The reasons for the
`variability between individuals in their. susceptibility
`to blockade of ovulation are not understood.
`These studies were completed using non-invasive
`
`l U methods with fewer than 20 animals. In practice
`
`such information is unobtainable in terminal experi(cid:173)
`ments. For example, it is estimated that detection of
`myometrial shrinkage at the same time as endometrial
`growth by terminal methods at the same significance
`level would have required over 750 animals. Thus,
`MRI may be a particularly sensitive and powerful
`tool for clinical studies in endocrine pharmacology.
`The clinical usefulness of ICI 182,780 remains to be
`determined, but its effect in a case of spontaneous
`adenomyosis in a monkey has been described
`(Waterton er al. 1993).
`
`ACKNOWLEDGEMENTS
`
`We thank S. A. Breen, M. Horrocks, D. Miller,
`J. S. W. Morrell, J. B. Larcombe-McDouall, D. Priest
`and J. Tattershall for expert technical assistance.
`
`REFERENCES
`
`Andreyko, J. L., Blumenfeld, Z., Marshall, L.A., Monroe, S. E.,
`Hricak, H. & Jaffe, R. B. (1988). Use of an agonistic analog
`of gonadotropin-re1easing hormone (nafarelin) to treat
`leiomyomas: assessment by magnetic resonance imaging.
`American Journal of Obstetrics and Gynecology 158, 903-910.
`Bartoli, J. M., Moulin, G., DeLannoy, L., Chagnaud, C. &
`Kasbarian, M . (1991). The normal uterus on magnetic
`resonance imaging and variations associated with the
`hormonal state. Surgical-Radiologic Anatomy 13, 213-220.
`Demas, B. E., Hricak, H. & Jaffe, R . B. (1986). Uterine
`imaging: effects of hormonal stimulation. Radiology 159,
`123-126.
`Dukes, M ., Miller, D ., Wakeling, A. E. & Waterton, J. C.
`(1992). Antiuterotrophic effects of a pure antioestrogen, ICI
`182,780: magnetic resonance imaging of the uterus in
`ovariectomiu:d monkeys. Journal of Endocrinology 135,
`239-247.
`Haynor, D . R ., Mack, L. A., Soules, M . R .• Shuman, W. P.,
`Montana, M . A. & Moss, A. A. (1986). Changing appearance
`of the normal uterus during the menstrual cycle. Radiology
`161, 459-462.
`McCarthy, S., Tauber, C. & Gore, J. (1986). Female pelvic
`anatomy: MR assessment of variations during the menstrual
`cycle and with use of oral contraceptives. Radiology 160,
`119-123.
`Wakeling, A. E. Dukes, M. &. Bowler, J. (1991). A potent
`specific pure antioestrogen with clinical potential. Cancer
`Research 51, 3867- 3873.
`Waterton, J. C., Breen, S. A., Dukes, M ., Horrocks, M. &
`Wadsworth, P. (1993). A case of adenomyosis diagnosed by
`magnetic resonance imaging and treated with the novel pure
`anti~trogen ICI I 82780. Laboratory Animal Science (In
`Press.)
`Watcrton, J . C., Larcombe-McDouall, J . B. &. Miller, D. ( 1992).
`Quantitative MRI of the prostate and uterus in monkeys.
`Magnetic Resonance in Medicine 28, 84-91 .
`Waterton, J. C., Miller, D., Dukes, M . & Morrell, J. S. W.
`(1991). Oblique NMR imaging of the uterus in macaques:
`uterine response to estrogen stimulation. Magnetic Resonance
`in Medicine 20, 228-239.
`Wiczyk, H. P., Janus, C. L., Richards, C. J., Graf, M . J.,
`Gendal, E . S., Rabinowitz, J. G . &. Laufer, N . (1988).
`Comparison of magnetic resonance imaging and ultrasound jn
`evaluating follicular and endomctrial development throughout
`the normal cycle. Fertility and Sterility 49, 969-975.
`Zawin, M ., McCarthy, S., Scoutt, L., Lange, R., Levy, G ., Vulte,
`J. & Comite, F. (1990). Monitoring therapy with a
`gonadotropin-rcleasing hormone analog: utility of MR
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`
`Journal of Endocrinology (1993) 138, 203-209
`
`MYLAN PHARMS. INC. EXHIBIT 1026 PAGE 7
`
`

`
`210 M. DUKES and others
`
`· Effects of IC/ 182,780 in adult menstruating monkeys
`
`DESCRIPTION OF PLATE
`
`Magnetic resonance (MR) images of the monkey uterus.
`Each illustration is a detail, representing 3·4 x 3·4 cm,
`from one slice of the eight recorded at each measurement.
`The appearance at mid-corpus uteri is shown as follows.
`Top row: images obtained at day 3 (Fig. 1), day 16 (Fig.
`2) and day 24 (Fig. 3) of an untreated menstrual cycle.
`Second row: images obtained at day 3 (Fig. 4), day 15
`(Fig. S) and day 24 (Fig. 6) in the same animal during a
`cycle in which O· l mg !CI 182, 780/kg was injected daily
`from day 3. Third row: images obtained at day 2 (Fig. 7),
`day 16 (Fig. 8) and day 23 (Fig. 9) of an untreated
`menstrual cycle. Bottom row: images obtained at day 2
`
`(Fig. 10), day 16 (Fig. 11) and day 23 (Fig. 12) in the
`same animal during a cycle in which 4 mg IC! 182,780/kg
`sustained-release formulation was injected on day 2. The
`cycles shown in the top three rows were ovulatory. Four
`or five concentric zones of MR signal intensity arc seen:
`central signal void, menstrual haemorrhage (only during
`menstruation); high signal, endometrium; medium-low
`signal, myometrial junction zone; medium-high signal,
`myometrium; and no signal, peripheral adipose tissue.
`The area (often crescent-shaped) of high signal intensity
`seen at the left in Figs 7- 12 is urine in the bladder. An
`ovary is partially visible (bottom right) in Figs 7- 1 1.
`
`(_ )
`
`•
`
`Journal of Endocrinology (1993) 138, 203-209
`
`MYLAN PHARMS. INC. EXHIBIT 1026 PAGE 8
`
`

`
`l'l .. \ 'lE
`
`l;"(li·av 11j' !Cl /t'C. 780 i1r ru/11/1 1111•11.11ma1i11g 11m11key.1·
`
`~I. Du.:r:s and othl.!rs
`
`2
`
`5
`
`8
`
`11
`
`., ...
`
`4
`
`10
`
`6
`
`9
`
`12
`
`MYLAN PHARMS. INC. EXHIBIT 1026 PAGE 9